Biosensors are chemosensors with a biological detection system. This detection system consists of biologically active substances, such as enzymes, antibodies, lectins, hormone receptors, etc., which are immobilized on the surface of the sensor or in a thin layer located on it. In the detection process, a change is produced on the surface or in this layer of the sensor, by interaction with the gaseous or liquid medium to be characterized, which can be evaluated using electrical, optical, mechanical, acoustical or calorimetric measurement methods. In the case of equipment with electronic data acquisition and evaluation, the active surface or layer is directly coupled, as a signal emitter, with a signal transformer, called a transducer, which is connected with the evaluation electronics for this purpose.
The reliability of the entire sensor depends on the assignability and reproducibility of the signals generated in the sensitive layer of the biosensor. This means that the layer must demonstrate not only high selectivity and sensitivity, but also a function that is free of hysteresis and drift, as well as chemical and biological stability and contamination resistance. For technical use, in particular, ease of operation, easy integration and the lowest possible measurement/regeneration time requirement are required, as well as great long-term stability. In addition, the production of the layer--according to methods which are efficient in terms of production technology and can be automated--should be as simple, reproducible and inexpensive as possible, and be such that it can be integrated into the production process for sensor production.
Until now, only-such biosensors which are based on enzymatic reactions have achieved any practical importance. In these reactions the circumstance is used that products which can easily be detected, such as H.sup.+, O.sub.2, H.sub.2 O.sub.2, CO.sub.2 and NH.sub.3, are formed or consumed. With regard to selectivity and sensitivity, the enzymatic reactions fully meet the requirements. But a difficulty exists in immobilizing the enzymes--without loss of activity--in as thin a detection layer as possible, in such a way that they are easily accessible for the substances to be detected, and are resistant to poisoning as well as biochemical pollutants, and remain functionally stable for as long as possible.
For the immobilization of enzymes, the following methods have been known:
adsorption on carrier surfaces PA1 ionic binding to carrier surfaces PA1 covalent binding to carrier surfaces PA1 absorption in polymer layers PA1 inclusion in a polymer lattice (matrix sheathing, microencapsulation) PA1 inclusion by sheathing with a membrane (macroencapsulation) PA1 cross-linking or copolymerization with difunctional or polyfunctional monomers. PA1 Z=vinyl group or photopolymerizable remainder with 8 to 40 C atoms, which can be obtained by addition of a photopolymerizable compound to a remainder E located at the siloxane chain, and subsequent addition of an aliphatic, cycloaliphatic or aromatic monoisocyanate or monoisothiocyanate with 2 to 10 C atoms to the secondary OH group formed upon opening of the epoxide ring, PA1 R.sup.1 =alkyl with 1 to 4 C atoms or phenyl, PA1 R.sup.2 =R.sup.1, E or Z, PA1 x=50 to 1000, y=10 to 300, z=3 to 8; PA1 x is preferably about 3 to 10 times y. PA1 Immobilization of all biochemical substances which have reactive NH, OH, SH or COOH groups at their periphery is made possible. PA1 The layers which have the immobilized biochemical substances can also be stored dry and under non-sterile conditions, without any damage to these substances. PA1 Immobilization of the biochemical substances takes place under very mild conditions, in aqueous solution and in the absence of reactive components with a low molecular weight; in this way losses, for example as the result of enzyme denaturing, are avoided. PA1 A relatively small number of polymer materials with great chemical and thermal stability, which can be produced on a large technical scale and which are therefore accessible at low cost, is used for immobilization of a large number of different types of biochemical substances and for different sensor types. PA1 The production and cross-linking of the layers, as well as their structuring, if necessary, can be carried out according to planar technology, i.e. in technically simple, reproducible and low-cost manner, and so as to be integrated into the sensor production. PA1 Immobilization of the biochemical substances can take place independent of the layer production, depending on the need and intended use, if necessary not until just before use, to be carried out by the user. PA1 Desorption, migration and extraction losses are avoided by chemical anchoring of the biochemical substances in the polymer matrix. PA1 By the formation of covalent bonds between the peripheral NH, OH, SH and COOH groups of the biochemical substances and the very soft and flexible sheathing polymer material, the substances, some of which are very sensitive, for example enzymes, are given great functional and long-term stability. PA1 Because of the possibility of the production of very thin layers (&lt;&lt;1 .mu.m), very short sensor response times can be achieved. PA1 Miniaturization and integration of the detection systems into microelectronic circuits, for example for the production of ISFETs and ENFETs, can be achieved without problems. PA1 The selective detection systems are basically suitable for all sensor measurement arrangements.
However, as is evident from the extensive literature on the immobilization of enzymes, all of these methods have disadvantages, which make them appear unattractive for industrial sensor production (see, for example: W. Hartmeier, "Immobilisierte Biokatalysatoren" ["Immobilized Biocatalysts"], Springer-Verlag Berlin, Heidelberg 1986, pages 23 to 51, as well as J. Woodward, "Immobilised cells and enzymes", IRL Press, Oxford, Washington D.C., 1985, pages 3 to 54).
Thus, adsorption and ionic binding of enzymes at the surface results in relatively unstable systems with a limited range of use: Changes in the pH and the ion intensity of solutions in contact with it, or the presence of other substances, already result in displacement of the surface-bound enzyme and thus to activity losses of the detection system. Also, in the case of absorption in polymer layers, with plasticized polyvinyl chloride being used in the predominant number of cases (see, for example: "Sensors and Actuators", Vol. 18 (1989), pages 329 to 336, and "Ber. Bunsenges. Phys. Chem." ["Reports of the Bunsen Society for Physical Chemistry"], Vol. 92 (1988), pages 1423 to 1426), relatively unstable systems are obtained: migration and extraction of the enzymes result in a constant decrease in activity (drift) and limit the lifetime of the sensor.
Significantly more stable systems are achieved if the enzymes are covalently bound to a carrier surface, made insoluble via cross-linking or copolymerization, or are immobilized by microencapsulation or macroencapsulation. For the formation of covalent bonds and for cross-linking, free amino, carboxyl, hydroxyl and mercapto groups are available on the part of the enzymes. Both inorganic materials, such as glass, and natural and synthetic organic polymers can be used as the carrier material. A prerequisite in this connection is that the carrier materials contain reactive groups, such as isocyanate, isothiocyanate, acid chloride and epoxy groups. Less reactive groups can be activated, for example carboxyl groups can be activated using the carbodiimide or azide method, hydroxyl groups can be activated using the bromine cyan method, and amino groups can be activated using the isothiocyanate or azo method. It was possible, particularly on the basis of acrylic acid and methacrylic acid derivatives, to produce numerous reactive copolymers with dinitrofluorophenyl, isothiocyanate, oxirane or acid anhydride groups. Polyacrylamides with oxirane groups as well as modified copolymers on the basis of vinyl acetate and divinyl ethylene urea with oxirane groups are commercially available, for example.
Immobilization by cross-linking or by copolymerization represent special forms of covalent binding. In these methods, the formation of covalent bonds takes place between the enzyme molecules and difunctional or polyfunctional monomers, such as glutardialdehyde, or, in the case of copolymerization, additionally between the enzyme molecules and a polymerizing substance. In this manner, insoluble aggregates with a high molecular weight are formed. Cross-linking is generally used as an immobilization method in combination with one of the other methods, for example in combination with adsorption or absorption. Here, the enzyme molecules are first adsorbed on the surface of the carrier, or are absorbed in a layer located on it, and subsequently cross-linked.
A significant disadvantage of immobilization by covalent binding is the great stress on the biocatalysts connected with it. The immobilization procedures that are necessary, some of which are rough, in which a strong change in the pH occurs, organic solvents have to be used or reaction with reactive substances with a low molecular weight takes place, almost always lead to strong conformation changes and thus to activity losses of enzymes bound in such manner.
In immobilization by inclusion, i.e. microencapsulation or macroencapsulation, the enzymes themselves are not made insoluble, rather their reaction range is limited by semipermeable polymers or polymer layers. A prerequisite for the ability of enzymes sheathed in this manner to function is that substrates and products can pass through the sheathing substance, while the enzymes themselves have to be held back. In addition to natural polymers, such as alginate, carrageenan, pectin, agar and gelatin, which are, however, too large-meshed for permanent immobilization of enzymes, synthetic polymers, such as polyacrylamide, are particularly used for matrix sheathing. Polyamides, polyurethanes, polyesters and polyureas, for example, are used for encapsulation. The inclusion method has the disadvantage that relatively thick layers with long sensor response times are formed.
In the methods described, immobilization of the enzymes is carried out by hand in most cases, which is relatively slow, expensive and not very reproducible, and is counter to integration into modern production processes. In view of the advantages which enzyme sensors on an FET basis (ENFETs) would be able to offer, attempts have been made in recent years to include enzyme immobilization into the planar technology in the production of integrated circuits. Thus, for example, the production and direct photo-structuring of layers based on polyvinyl alcohol which contain enzymes and can be photo-cross-linked has been described ("Proc. 3rd Int. Conf. Solid State Sensors and Actuators (Transducers '85)" Jun. 11-14, 1985, pages 148 to 151). For the purpose stated, it is also known to use photosensitive polyvinyl pyrrolidone "(IEEE Trans Electron Devices" Vol ED-36 (1989), pages 1303 to 1310). According to this method, structures which exactly cover the gates of the FETs can be produced on wafers. However, this method has the great disadvantage that the enzymes are at least partially inactivated during UV irradiation.
It is also known to utilize enzyme inactivation by means of UV radiation, in that first a layer of acetyl cellulose containing an enzyme is produced, the enzyme is cross-linked with glutardialdehyde in this layer, and subsequently it is irradiated through a mask in such a way that the gate coverings are shaded and therefore remain active, while the remaining areas are inactivated ("Chemical Economy & Engineering Review", Vol. 17 (1985), No. 7-8, pages 22 to 27). The inactivated layer remains on the sensor, which proves to be a disadvantage for further insulation and packaging of the sensor required for its use.
The lift-off technique has also been described ("Sensors and Actuators" Vol 13 (1988), pages 165 to 172). In this method, a photoresist is structured in such a way that only the gate surfaces remain free. The enzyme is then applied to this, together with glutardialdehyde, and cross-linked; the photo varnish is removed with acetone and ultrasound, using the lift-off technique. Here again, it is impossible to avoid at least partial denaturing of the enzyme.